Histological Features of the Testis in the Long-beaked Common Dolphin from Korean Waters

1Depaertment of Veterinary Anatomy, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea 2Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine,Chungbuk National University, Cheongju 361-763, Republic of Korea3Cetacean Research Institute, National Fisheries Research & Development Institute, Ulsan 680-050, Republic of Korea

(접수: 2013. 2. 25/ 심사: 2013. 2. 25/ 채택: 2013. 3. 5)

Abstract

The present study examined the histological characteristics of adult testis in the long-beaked common dolphin(Delphinus capensis) from Korean waters and the localization of DEAD-box polypeptide 4 (DDX4; a germ cell marker)and vimentin (a Sertoli cell marker) expression in the dolphin testis compared with that in terrestrial mammals, includingdogs and rats. The seminiferous tubules of dolphin testis have very small or completely closed lumens, andspermatogenic cells and Sertoli cells within the tubules cannot be differentiated. Immunohistochemical analysis showedthat, in the dolphin testis, DDX4- and vimentin-positive cells were scattered extensively within the tubule, whereasin the dog and rat testis, DDX4 immunoreactivity was localized in spermatogenic cells of the adluminal compartment,and vimentin immunoreactivity was localized in Sertoli cells of the basal compartment in the seminiferous epithelium.These results suggest that the histological characteristics of the seminiferous tubules in the dolphin testis differ fromthose of terrestrial species.

INTRODUCTION

The long-beaked common dolphin (Delphinus capensis) is a species of the common dolphin genus Delphinus within the Delphinidae family (Natoli et al., 2005). There are two species of Delphinus: the short-beaked common dolphin (D. delphis) and the long-beaked common dolphin (Wilson and Reeder, 2005). The long-beaked common dolphin has been found in a more restricted area than has the short-beaked common dolphin, namely the western and southern Africa, western South America, central California to central Mexico, coastal Peru, and areas around Japan, Korea and Taiwan (Wilson and Reeder, 2005). Adults range from 1.9 to 2.5 m in length, and weigh 80 to 150 kg, and have a long, thin rostrum (Shirihai, 2006). They have a gestation period of 10 to 11 months, and the typical interbirth interval ranges from 1 to 3 years (Shirihai, 2006).

The histological characteristics of testes may prove highly informative in the analysis of the reproductive cycle and development. Classification of animals according to their stage of sexual development was undertaken using gonadal morphology and the distribution of cytoskeletal proteins in the male harbor porpoise Phocoena phocoena in the eastern North Atlantic (Holt et al., 2004). The testes of male short-beaked common dolphins in the western North Atlantic were characterized by pronounced seasonal changes in both morphology and histology (Westgate and Read, 2007). In a recent paper, histological data showed distinctive features of the seminiferous tubules in the male franciscana dolphin Pontoporia blainvillei in Argentine waters (Panebianco et al., 2012). However, little information is available about the morphology and histology of the long-beaked common dolphin testes.

In the present study, we examined the histological characteristics of the adult testis in the long-beaked common dolphin D. capensis from Korean waters and the localization of DEADbox polypeptide 4 (DDX4; a germ cell marker) and vimentin (a Sertoli cell marker) expression in the dolphin testis compared with that in terrestrial mammals, including dogs and rats.

MATERIALS AND METHODS

1. Animals and Tissue Preparation

Adult testis samples (left testis 2,442 g; right testis 2,744 g) were obtained from a long-beaked common dolphin (length 2.64 m) that had become caught in a commercial fishing set net on May 5, 2012. The samples were kept frozen until they were processed. After thawing, the adult testes were fixed in 10% buffered formalin, and embedded in paraffin wax. Fivemicrometer-thick sections were mounted on glass slides and stained with hematoxylin and eosin (H&E) following a routine protocol.

3. Immunohistochemistry

Five-micrometer-thick sections were deparaffinized, hydrated, and incubated overnight at 4℃ with rabbit anti-DDX4/MVH (1:500 dilution) and mouse anti-vimentin (1:400 dilution) antibodies in phosphate-buffered saline containing 0.1% Tween 20 (PBS-T, pH 7.4). The sections were then incubated with biotinylated goat anti-rabbit IgG and horse anti-mouse IgG (Vector ABC Elite Kit; Vector Laboratories, Burlingame, CA, USA) in PBS-T for 45 min at room temperature (RT). Immunoreactivity was detected by incubation for 45 min with the avidinbiotin peroxidase complex (Vector ABC Elite Kit; Vector Laboratories) prepared according to the manufacturer’s instructions. The peroxidase reaction was visualized using a diaminobenzidine substrate kit (SK-4100; Vector Laboratories). As a control, the primary antibodies were omitted for a few test sections in each experiment. The sections were counterstained with Harris hematoxylin before being mounted.

4. Double-Immunofluorescence

The separate distribution of DDX4 and vimentin was examined by double-immunofluorescence labeling in the same sections. In brief, sections were incubated overnight at 4℃ with mouse rabbit anti-DDX4/MVH antibody (1:500 dilution) in PBS-T and were then treated with TRITC-conjugated antirabbit IgG (1:50 dilution) in PBS-T for 1 h at RT. Next, the sections were incubated for 2 h at RT with mouse anti -vimentin (1:400 dilution) in PBS-T followed by FITC-conjugated anti-mouse IgG (1:50 dilution) in PBS-T for 1 h at RT. The sections were counterstained during incubation for 30 min at room temperature with 5 μg/ml of 4’,6-diamidino-2-phenylindole 2HCl (DAPI; Thermo Fisher Scientific, Rockford, IL, USA) in PBS. The double-immunofluorescencestained sections were examined using a BX-40 fluorescence microscope (Olympus, Tokyo, Japan) equipped with an eXcope X3 digital camera (DIXI Optics, Daejeon, South Korea).

RESULTS

1. Histological Analysis of the Long-beak Common Dolphin Testis

We first performed H&E staining to examine the characteristic features of mature long-beaked common dolphin testis. The transverse section of the dolphin testis showed stretched seminiferous tubules with large (>200 μM) tubule diameters (Fig. 1A) and very small or completely closed lumens (Fig. 1A and C). Furthermore, the testis sections showed evidence of spermatogenesis: spermatozoa were present in the tubules (Fig. 1C), and mature sperm were present in the epididymis (Fig. 1B and D). However, it was difficult to differentiate between spermatogenic cells and Sertoli cells in the sections with H&E staining.

2. Localization of DDX4 and Vimentin Expression in the Long-beak Common Dolphin: Comparison with Terrestrial Mammals

To clarify the localization of spermatogenic cells and Sertoli cells in mature long-beaked common dolphin testis, we examined the immunoreactivities of DDX4 and vimentin in the tubules and the expressions were compared with those of dog and rat testis.

In the dolphin testis, numerous DDX4-positive cells were scattered extensively in the seminiferous tubule (Fig. 2A). However, in the dog and rat testis, DDX4 immunoreactivity was localized in spermatogenic cells, especially spermatocytes and spermatids in the adluminal compartment of the seminiferous epithelium (Fig. 2B and C).

Fig. 2. Immunohistochemical analysis of DDX4 in the testis of the adult long-beaked common dolphin, dog and rat. (A) Representative image of DDX4 immunoreactivity in the dolphin testis. DDX4-immunoreactive cells were localized extensively in the testis. (B and C) Representative images of DDX4 immunoreactivity in the dog and rat testis. DDX4-immunoreactive cells were localized exclusively in adluminal compartments of the seminiferous epithelium. Sections were counterstained with Harris’ hematoxylin. Scale bars = 70 μm.

Several vimentin-positive cells in the dolphin testis were detected extensively in the seminiferous tubules (Fig. 3A), whereas the positive cells in the dog and rat testis were detected mainly in the basal compartment of the seminiferous epithelium (Fig. 3B and C).

Fig. 3. Immunohistochemical analysis of vimentin in the testis of the adult long-beaked common dolphin, dog and rat. (A) Representative image of vimentin immunoreactivity in the dolphin testis. Vimentin-immunoreactive cells were localized extensively in the testis. (B and C) Representative images of vimentin immunoreactivity in the dog and rat testis. Vimentin-immunoreactive cells were localized exclusively in basal compartments of the seminiferous epithelium. Sections were counterstained with Harris’ hematoxylin. Scale bars = 70 μm.

To clarify the difference in distribution of DDX4 and vimentin expression in the seminiferous tubules of the dolphin testis, doubleimmunofluorescence was performed in the testis. The results showed that DDX4 immunoreactivity (spermatogenic cells) did not overlap with vimentin (Sertoli cells) in the tubules (Fig. 4).

DISCUSSION

The present study demonstrated the histological features of the testis of the adult long-beaked common dolphin and compared the localization of DDX4 and vimentin immunoreactivity in dolphin testis with that in dog and rat testis.

It is generally accepted that males are classified as immature or mature based on testis morphology and histology. Mature dolphins have histological characteristics of spermatozoa in the tubules and/or mature sperm in the epididymis, and larger (>100 μM) tubule diameters (Westgate and Read, 2007). In common dolphins, seasonal changes in testis activity can confound this type of analysis, so the ages and standard lengths of all individuals in the assessment were considered (Westgate and Read, 2007). Moreover, both morphology and histology of the testes of short-beaked common dolphins from the western North Atlantic pronounced seasonal changes (Westgate and Read, 2007). The seasonal increase in testis size was found to begin in early May, and the testis mass reached a maximum in the middle of July (4,049.6 ± 1,317.4 g); however, the testes were fully regressed by October (802.9 ± 455.5 g) (Westgate and Read, 2007). In the present study, the longbeaked common dolphin measured 2.641 m in length, and the left testis weighed 2,442 g, and the right testis weighed 2,744 g. Histological analysis of the transverse section of the dolphin testis revealed large seminiferous tubules (tubule diameters, >200 μM) and spermatozoa present within the tubules. Thus, the long-beaked common dolphin inadvertently caught in May from Korean waters was found to be a mature animal possibly experiencing a seasonal increase in testis size and seminiferous tubule diameter.

DDX4, also called VASA or mouse vasa homolog (MVH), has an important role in spermatogenesis and is specifically expressed in the cytoplasm of spermatogenic cells (Toyooka et al., 2000; Chuma et al., 2006). DDX4 has an essential role in meiosis during spermatogenesis, and is distributed uniformly in the cytoplasmic region from spermatogonia to leptotene spermatocytes (Noce et al., 2001). Consistent with previous studies, DDX4 immunoreactivity was present in spermatogenic cells of the seminiferous tubules (especially those in the adluminal compartment of the epithelium). However, unlike the case in dog and rat testis, DDX4-positive cells were scattered extensively in the seminiferous tubules of the dolphin testis. Therefore, it is suggested that the localization of DDX4-positive spermatogenic cells in the dolphin testis is different from that in dog and rat.

Vimentin intermediate filament also plays a pivotal role in the maintenance of spermatogenesis in the testis and is regarded as a useful marker for Sertoli cells (Paranko et al., 1986; Kopecky et al., 2005). Several studies have shown region-specific vimentin expression in the Sertoli cells of the testis of many species (Mali et al., 1987; Steger et al., 1996; Peters et al., 2001). The present study also compared the vimentin expression pattern in dolphin testis with that in dog and rat testis. In dog and rat testis, vimentin immunoreactivity was expressed mainly in the Sertoli cells (especially the basal compartment of the seminiferous epithelium). However, the long-beaked common dolphin testis showed scattered expression of vimentin in the seminiferous tubules. Therefore, it is suggested that in the dolphin testis, in contrast to dog and rat testis, the localization of vimentin immunoreactivity was present diffusely in both the basal or adluminal compartment.

In conclusion, in the present study, an adult long-beaked common dolphin was characterized in terms of both the morphology and histology of its testes. The localization of DDX4 and vimentin immunoreactivities differed from that in dog and rat testis, suggesting that the different localization may be implicated in the seasonal change in testis activity of the adult dolphin. However, further studies will be needed to clarify the relationship between the localization of DDX4 and vimentin immunoreactivity and testis activity depending on the seasonal change in the dolphin testis.